Glutathione is a tripeptide, L-.gamma.-glutamyl-L-cysteinylglycine, present in high concentrations in most cell types. By virtue of its reactive sulfhydryl group, glutathione is able to donate a hydrogen ion and unpaired electron and neutralize peroxides and free radicals (Meister, A., Nutrition Reviews 42:397-410 (1984); Meister, A., J. Biol. Chem. 263:17205-17208 (1988); Kosower et al., Int. Rev. Cytology 54:109-160 (1978)).
Experimental data demonstrate that glutathione and its redox system enzymes, glutathione peroxidase and reductase, provide a widespread and essential protection system from both endogenous and exogenous oxidative assault. In numerous cell types, normal or enhanced levels of glutathione are protective against cellular injury induced by a variety of different agents (Harlan et al., J. Clin. Invest. 73:706-713 (1984); Roos et al., Agents and Actions 10:528-535 (1980); Weinberg et al., J. Clin. Invest. 80:1446-1454 (1987); Babson et al., Biochem. Pharm. 30:2299-2304 (1981); Lash et al., Proc. Natl. Acad. Sci. USA 83:4641-4645 (1986); Szabo et al., Science 214:200-202 (1981)). Conversely, depletion of glutathione has been demonstrated to sensitize tissues to increased oxidative injury by various stresses (Deneke et al., J. Appl. Physiol. 58:571-574 (1985); Davis et al., Current Surgery 45:392-395 (1988); Chen et al., Biochem. Biophys. Res. Comm. 151:844-850 (1988)).
Glutathione synthesis is directed by the sequential activities of .gamma.-glutamylcysteine synthetase (GGCS) and glutathione synthetase. GGCS is the rate limiting enzyme, and is feedback inhibited by intracellular glutathione levels. In addition, the rate of synthesis can be regulated by substrate availability. It has been reported that cysteine is rate-limiting for glutathione synthesis. (Meister, A., Nutrition Reviews 42:397-410 (1984); Richman et al., J. Biol. Chem. 250:1422-1426 (1975)).
Degradation of glutathione is dependent upon .gamma.-glutamyl transpeptidase (GGTP), a membrane bound enzyme, which catalyzes the transfer of the .gamma.-glutamyl group of glutathione to an acceptor molecule, either an amino acid or water, to form a .gamma.-glutamyl amino acid or glutamate respectively. The cysteine-glycine moiety of the degraded glutathione is quickly broken down by a dipeptidase and each amino acid is absorbed intracellularly. The .gamma.-glutamy amino acid is translocated into the cell and acted upon by .gamma.-glutamyl cyclotransferase to form the free amino acid and oxoproline. Oxoproline (pyroglutamic acid) is converted to glutamate by 5-oxoprolinase. Glutamate can then be used for glutathione synthesis to complete the cycle (Meister, A., Nutrition Reviews 42:397-410 (1984)).
The .gamma.-glutamyl cycle has been shown to exist in many cell types, but its precise physiologic function is not well understood. It has been proposed that the formation of .gamma.-glutamyl amino acid constitutes one form of an amino acid transport mechanism (Griffith et al., Proc. Natl. Acad. Sci. USA 76:6319-6322 (1979)). However, others have noted that under physiologic conditions, the hydrolysis of the .gamma.-glutamyl complex with the formation of glutamate is the dominant reaction (McIntyre et al., Int. J. Biochem. 12:545-551 (1980); Cook et al., Biochim. Biophys. Acta 884:207-210 (1986)).
It has been reported that toxic doses of endotoxin in mice significantly decreased the concentration of non-protein bound sulfhydryl groups, of which glutathione comprised ninety percent. It was demonstrated that scalding, hind leg ligation, endotoxin administration, exposure to cold, tumbling trauma, and severe hemorrhage all resulted in significant decreases in liver glutathione levels. The mechanism of this depletion and its significance were not understood (Beck et al., Proc. Soc. Expt. Biol. 81:291-294 (1952); Beck et al., Proc. Soc. Expt. Biol. 86:823-827 (1954)).
Following the observations of Beck et al., other investigators examined the effects of glutathione in a number of animal shock models. The exogenous administration of glutathione to animals in endotoxic shock, (Szymanski et al., Proc. Soc. Expt. Biol. 129:966-968 (1968); Sumida et al., Jap. Circ. J. 45:1364-1368 (1981); Kosugi et al., "New Approaches to Shock Therapy: Reduced GSH," in Molecular Aspects of Shock and Trauma, A. M. Lefer, ed., Alan R. Liss, Inc., New York (1983)), hemorrhagic shock (Horejsi et al., Folia Haematol. 86:220-225 (1966); Yamada, H., Jap. J. Anesth. 26:640-645 (1977)), and cardiogenic shock (Galvin et al., Am. J. Physiol. 235:H657-H663 (1978)), significantly attenuated tissue injury and improved survival. In addition, recent evidence has demonstrated that tumor necrosis factor may induce cell damage by oxidative injury (Watanabe et al., Immunopharm. Immunotox. 10:109-116 (1988); Matthews et al., Immunology 62:153-155 (1987)), and that in rats, depletion of glutathione levels enhanced mortality to previously non-lethal doses of tumor necrosis factor (Zimmerman et al., J. Immunology 142:1405-1409 (1989)).
Intestinal mucosal levels of glutathione have also been shown to decrease significantly after 24 to 48 hours of starvation (Ogasawara et al., Res. Exp. Med. 189:195-204 (1989); Siegers et al., Pharmacology 39:121-128 (1989)). Erythrocyte glutathione levels do not change during this period of starvation, consistent with the longer, four day, intracellular half-life (Cho et al., J. Nutr. 111:914-922 (1981)).
Radiation therapy is a regional form of treatment for control of localized cancers. Success of radiotherapy depends upon the production of free radicals by the ionizing events following irradiation. The resulting free radicals and oxidizing agents produce DNA strand breaks and other damage to DNA molecules in the localized cancer. However, radiotherapy is associated with accompanying damage to normal tissues as well, and damage to normal tissues increases with the size of the tumor. Prevention or reduction of the oxidative damage to normal tissue would be of benefit to a patient receiving radiotherapy.
Many therapeutic substances can cause liver damage by virtue of the production of oxidative metabolites. Acetaminophen (paracetamol) is a commonly used over-the-counter analgesic preparation, and a frequent cause of poisoning. A metabolic route of acetaminophen is a cytochrome P-450 catalyzed activation which results in the formation of a reactive metabolite that binds to cellular nucleophiles, particularly reduced glutathione.
Another common substance which can cause oxidative damage to the liver is acrolein, a metabolite of the widely used anticancer drug cyclophosphamide. Acrolein binds to cellular sulfhydryls and can deplete intracellular glutathione, leading to cell death. (Dawson, J. R. et al., Arch. Toxicol. 55:11-15 (1984)). The early clinical manifestations of cyclophosphamide toxicity include hemorrhagic cystitis, sterility, and alopecia. (Izard, C. et al.. Mutation Research 47:115-138 (1978)).
Compounds capable of causing oxidative damage are not limited to intentionally administered pharmaceuticals. Paraquat is an herbicide which has toxic effects on most organs including the lungs, liver, heart, gastrointestinal tract and kidneys. Paraquat undergoes a redox cycling reaction which can lead to the production of reactive oxygen species, including hydrogen peroxide and the superoxide radical. (Dawson, J. R. et al., Mutation Research 47:115-138 (1978)).
N-acetylcysteine has a protective effect against the toxicity of acetaminophen, acrolein and paraquat in isolated hepatocytes. Acting as a precursor for glutathione, N-acetylcysteine decreased the toxicity of paraquat co-incubated with hepatocytes. (Dawson, J. R. et al., Arch. Tox. 55:11-15 (1984)). N-acetylcysteine is currently a clinical treatment of choice for patients who have ingested excess amounts of acetaminophen. However, N-acetylcysteine is not approved for intravenous use in the United States, and is thus not available for patients presenting with compromised gastrointestinal function.
In cases of acetaminophen overdose, depletion of intracellular glutathione can lead to cell death and liver damage. (Dawson, J. R. et al., Arch. Tox. 55:11-15 (1984)). In Great Britain alone, over 150 people die each year as a result of acetaminophen poisoning. (Meredith, T. J. et al., Br. Med. J. 293:345-346 (1986)). In a study of 100 patients with acetaminophen-induced liver failure, a 37% mortality was observed despite administration of the currently used antidote, acetylcysteine. Mortality was 58% among patients not receiving the antidote. (Harrison, P. M. et al., The Lancet:1572-1574 (Jun. 30, 1990.)) Thus, although the currently used treatment achieves some reduction in mortality, a more effective treatment is needed to further reduce the mortality rate.
Acetaminophen, cyclophosphamide, and other drugs that can be metabolized to toxic derivatives, are often administered to patients already under significant physical stress due to illness and lack of nutrition. In these patients, the hepatic stores of glutathione may have fallen below normal levels, lowering the detoxifying capability of the liver. The effect of starvation on tissue glutathione levels is therefore important in view of the diminished nutritional status of patients receiving anti-cancer drugs or other potent pharmaceutical agents.
Hepatic glutathione levels fall approximately 50% within 24 to 48 hours of starvation or low protein diet (Leaf et al., Biochem. J. 41:280-287 (1947); Cho et al., J. Nutr. 111:914-922 (1981); Strubelt et al., Toxic. Appl. Pharm. 60:66-77 (1981)). This is consistent with the short half life of liver glutathione of approximately 4 hours. With refeeding, hepatic levels of glutathione return to normal within 24 hours. Exogenous administration of glutathione, either parenterally or intraperitoneally, is relatively ineffective in enhancing tissue levels (Anderson et al.. Arch. Biochem. Biophys. 239:538-548 (1985)). Plasma glutathione is rapidly metabolized, and most tissues are unable to transport large amounts of intact exogenous glutathione. The small amounts of glutathione present in plasma are due primarily to rapid hepatic synthesis and release, and to rapid renal degradation.
Although plasma levels of glutathione are 100-500 times lower than intracellular levels, a significant amount of glutathione is able to circulate because of its rapid flux (Griffith et al., Proc. Natl. Acad. Sci. USA 76:5606-5610 (1979)). Hirota et al. have hypothesized that the release of plasma glutathione by the liver is important in the protection of cell membranes from oxidative damage. Shock-induced hepatic dysfunction may inhibit sufficient synthesis and release of plasma glutathione, enabling subsequent oxidative damage to occur (Hirota et al., Gastroenterolgy 97:853-859 (1989); Keller et al., Arch. Surg. 120:941-945 (1985)).
Patients unable to take in adequate nutrition are often treated with total parenteral nutrition formulas. Parenteral administration of pharmaceutical preparations is also appropriate for patients with gastrointestinal dysfunction. However, the commonly used antidote for acetaminophen overdose, N-acetylcysteine, is not approved for intravenous use in the United States. Thus, patients presenting with non-functional or dysfunctional gastrointestinal systems associated with acetaminophen overdose cannot be provided with N-acetylcysteine intravenously.
In view of the crucial role played by glutathione in detoxification of drug metabolites and in preventing peroxidation of cell components, a method for maintaining hepatic stores of glutathione, particularly during times of stress to the body, including chemotherapy, is needed.